Effects of notoginseng leaf triterpenes on small molecule metabolism after cerebral ischemia/reperfusion injury assessed using MALDI-MS imaging.

BACKGROUND: Notoginseng leaf triterpenes (PNGL) is believed to have neuroprotective effects via the inhibition of inflammatory response and neuronal apoptosis. However, its mechanisms underlying the anti-ischemia/reperfusion (I/R) injury effects on the regulation of small molecule metabolism in rat brain remains unclear. The purpose of this study was thus to explore the mechanisms of PNGL on the regulation of small molecule metabolism in rat brain after I/R injury using matrix-assisted laser desorption ionization-mass spectrometry imaging (MALDI-MSI). METHODS: As a model of in vivo cerebral I/R injury, male Sprague-Dawley (SD) rats were established with a middle cerebral artery occlusion/reperfusion (MCAO/R) model after PNGL administration with 40 mg·kg-1 through intraperitoneal injection (i.p.) for 7 days. We assessed the neurological behavior, regional cerebral blood flow (r CBF), neuron injury, and spatial distribution of metabolic small molecules. RESULTS: Our in vivo results suggested that PNGL increased cerebral blood flow and relieved neurological dysfunction. Furthermore, using MALDI-MSI, we demonstrated that PNGL regulated 16 endogenous small molecules implicated in metabolic networks including tricarboxylic acid (TCA) cycle, adenosine triphosphate (ATP) metabolism, malate-aspartate shuttle, metal ions, and antioxidants underwent noticeable changes after reperfusion for 24 h. CONCLUSIONS: PNGL is a novel cerebrovascular agent that can improve cerebral blood flow and attenuate adverse neurological disorders. The mechanisms are closely correlated with relative metabolic pathways, which offers insight into exploring new mechanisms in PNGL for the treatment of cerebral I/R injury.

Effects of Dl-3-n-butylphthalide on Cerebral Ischemia Infarction in Rat Model by Mass Spectrometry Imaging.

Dl-3-n-butylphthalide (NBP) is a drug that is used in the treatment of ischaemic stroke. However, to the best of our knowledge, there are no systematic studies investigating the effects of dl-3-n-butylphtalide on the brain metabolism of small molecules. In this study, we first investigated the effects of dl-3-n-butylphthalide on the spatial distribution of small molecules in the brains of rats with permanent middle cerebral artery occlusion (pMCAO) using matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) imaging. After pMCAO modelling or a sham operation, rats were given four mg/kg of dl-3-n-butylphthalide through the caudal vein or saline once a day for nine days. The degree of neurological deficit in rats was evaluated using the modified neurological severity score (mNSS). MALDI-TOF-MS imaging was used to observe the content and distribution of small molecules related to metabolism during focal cerebral ischaemia. Multiple reaction monitoring (MRM) mode with liquid chromatography tandem mass spectrometry (LC-MS/MS) was used to verify the results obtained from MALDI-TOF-MS imaging. These small molecules were found to be involved in glucose metabolism, ATP metabolism, the glutamate-glutamine cycle, malate aspartate shuttle, oxidative stress, and inorganic ion homeostasis. Of the 13 metabolites identified by MALDI-TOF-MS imaging, seven compounds, ATP, ADP, AMP, GMP, N-acetylaspartic acid, ascorbic acid and glutathione, were further validated by LC-MS/MS. Taken together, these results indicate that dl-3-n-butylphthalide significantly improved ATP metabolism, level of antioxidants, and sodium-potassium ion balance in a rat model of pMCAO.

PARK7 protein translocating into spermatozoa mitochondria in Chinese asthenozoospermia.

PARK7 (DJ1) is a multifunctional oxidative stress response protein that protects cells against reactive oxygen species (ROS) and mitochondrial damage. PARK7 defects are known to cause various physiological dysfunctions, including infertility. Asthenozoospermia (AS), i.e. low-motile spermatozoa in the ejaculate, is a common cause of human male infertility. In this study, we found that downregulation of PARK7 resulted in increased levels of lipid peroxide and ROS, decreased mitochondrial membrane potential, and reduced mitochondrial complex I enzyme activity in the spermatozoa from AS patients. Furthermore, it was observed that PARK7 was translocated into the mitochondria of damaged spermatozoa in AS. Finally, we examined the oxidative state of PARK7 and the results demonstrated the enhancement of oxidation, expressed by increased sulfonic acid residues, the highest form of oxidation, as the sperm motility decreased. Taken together, these results revealed that PARK7 deficiency may increase the oxidative stress damage to spermatozoa. Our present findings open new avenues of therapeutic intervention targeting PARK7 for the treatment of AS.

Protocatechualdehyde prevents methylglyoxal-induced mitochondrial dysfunction and AGEs-RAGE axis activation in human lens epithelial cells.

Methylglyoxal (MGO), a glucose derived dicarbonyl intermediate, is a major precursor of advanced glycation end products (AGEs) which have been linked to the development of diabetic cataract. Protocatechualdehyde (PCA), a phenolic acid compound, is found in the roots of Salvia miltiorrhiza. This study was to investigate the effect of PCA against MGO-induced cytotoxicity in human lens epithelial cells (SRA01/04 cells) and the possible involved molecular mechanism. The results showed that PCA alleviated MGO-induced mitochondrial dysfunction and apoptosis in SRA01/04 cells. Furthermore, PCA was capable of inhibiting MGO-mediated AGEs formation and blocking receptor of AGEs expression in SRA01/04 cells. It is concluded that PCA could be useful in attenuation of MGO-induced cell damage and the possible mechanism is involved in modulating AGEs-receptor of AGEs axis in human lens epithelial cells, which suggests that PCA has a potential protective effect on diabetic cataract.

Serum proteomic analysis reveals high frequency of haptoglobin deficiency and elevated thyroxine level in heroin addicts.

Heroin addiction is a chronic, complex disease, often accompanied by other concomitant disorders, which may encumber effective prevention and treatment. To explore the differences in expression profiles of serum proteins in control and heroin addicts, we used two-dimensional electrophoresis coupled to MALDI-TOF/TOF, and identified 4 proteins of interest. Following validation of the increase in serum transthyretin, we assessed serum levels of thyroid stimulating hormone (TSH), triiodothyronine (T3), and thyroxine (T4), and observed a robust increase in T4 in heroin addicts compared to controls. In addition, we performed haptoglobin (Hp) phenotyping, and showed that the frequency of Hp0 (serum devoid of haptoglobin) was significantly higher in heroin addicts. Altogether, these findings indicated that: (1) thyroid hormone imbalance is present in heroin addicts; (2) anhaptoglobinemia (Hp0) might a risk factor or a deleterious effect of heroin abuse.

Two xanthones from Swertia punicea with hepatoprotective activities in vitro and in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: Swertia punicea Hemsl. (Gentianaceae) is more commonly known as "Ganyan-cao" and used mainly as a traditional Chinese folk medicine for the treatment of acute bilious hepatitis, cholecystitis, fever, intoxification and jaundice. MATERIALS AND METHODS: The active hepatoprotective constituents of Swertia punicea were purified using various column chromatography techniques. The structures of two isolated compounds were determined on the basis of spectroscopic data interpretation such as NMR analysis. The hepatoprotective activities of isolated compounds were evaluated by using hepatotoxicity in vitro and dimethylnitrosamine-induced rat hepatic fibrosis in vivo, respectively. RESULTS: Two xanthones, 1, 7-dihydroxy-3, 4, 8-trimethoxyxanthone (1) and bellidifolin (2) were isolated from the stems of Swertia punicea. The compounds 1 and 2 exhibited notable hepatoprotective activities against carbon tetrachloride (CCl4) -induced HepG2 cell damage, and effectively alleviated the levels of aspartate transaminase (AST), lactate dehydrogenase (LDH), superoxide dismutase (SOD) and malonic dialdehyde (MDA) induced by CCl4 in a concentration-dependent manner. Co-treatment with compound 2 significantly increased the cell viability compared with N-acetyl-p-aminophenol (APAP) treatment. Compound 2 also alleviated APAP-induced hepatotoxicity by increasing glutathione (GSH) content and decreasing hydroxyl free radical (·OH) levels and reactive oxygen specises (ROS) production. In addition, the protective effect of compound 1 significantly alleviated DMN-induced liver inflammation and fibrosis. Oral administration of compound 1 recovered the reduction of albumin (ALB) and reversed the elevation of serum alanine transaminase (ALT), AST and total bilirubin (TBIL) in dimethylnitrosamine (DMN)-induced fibrotic rats. Severe oxidative stress induced in fibrotic rats was evidenced by a 1.5-fold elevation in MDA and a fall in the SOD activity, and treatment with compound 1 protected against these adverse effects. Recovery of rat liver tissue against DMN-induced hepatocellular necrosis, inflammatory changes and hepatic fibrosis by compound 1 is also confirmed by H&E and Masson stained histopathological evaluation of liver tissue. CONCLUSION: Two xanthones from Swertia punicea exhibited hepatoprotective activities in vitro (compounds 1 and 2) and in vivo (compound 1), respectively.

Myricitrin alleviates methylglyoxal-induced mitochondrial dysfunction and AGEs/RAGE/NF-κB pathway activation in SH-SY5Y cells.

Advanced glycation end products (AGEs) have been identified in age-related intracellular protein deposits of neurodegenerative diseases. Methylglyoxal (MGO), a dicarbonyl metabolite, is a major precursor of AGEs which have been linked to the development of neurodegenerative diseases. Myricitrin, a flavanoid isolated from the root bark of Myrica cerifera, attenuated 6-OHDA-induced mitochondrial dysfunction and had a potential anti-Parkinson's disease in our previous investigation. The aims of this study were to investigate the protective effects of myricitrin against MGO-induced injury in SH-SY5Y cells and also to look for the possible mechanisms. The results showed that exposure of SH-SY5Y cells to MGO caused decreases of cell viability, intracellular ATP, mitochondrial redox activity, and mitochondrial membrane potential and an increase in reactive oxygen species generation. However, these mitochondrial dysfunctions were alleviated by co-treatment with myricitrin. Additionally, myricitrin was capable of inhibiting AGEs formation, blocking RAGE expression, and inhibiting NF-κB activation and translocation triggered by MGO in SH-SY5Y cells. Our results suggest that myricitrin alleviates MGO-induced mitochondrial dysfunction, and the possible mechanism is through modulating the AGEs/RAGE/NF-κB pathway. In summary, myricitrin might offer a promising therapeutic strategy to reduce the neurotoxicity of reactive dicarbonyl compounds, providing a potential benefit agent with age-related neurodegenerative diseases.

Neuroprotective effects of protocatechuic aldehyde against neurotoxin-induced cellular and animal models of Parkinson's disease.

Protocatechuic aldehyde (PAL) has been reported to bind to DJ-1, a key protein involved in Parkinson's disease (PD), and exerts potential neuroprotective effects via DJ-1 in SH-SY5Y cells. In this study, we investigated the neuroprotective pharmacological effects of PAL against neurotoxin-induced cell and animal models of PD. In cellular models of PD, PAL markedly increased cell viability rates, mitochondrial oxidation-reduction activity and mitochondrial membrane potential, and reduced intracellular ROS levels to prevent neurotoxicity in PC12 cells. In animal models of PD, PAL reduced the apomorphine injection, caused turning in 6-OHDA treated rats, and increased the motor coordination and stride decreases in MPTP treated mice. Meanwhile, in an MPTP mouse model, PAL prevented a decrease of the contents of dopamine (DA) and its metabolites in the striatum and TH-positive dopaminergic neuron loss in the substantia nigra (SN). In addition, PAL increased the protein expression of DJ-1 and reduced the level of α-synuclein in the SN of MPTP lesioned mice. PAL also increased the spine density in hippocampal CA1 neurons. The current study demonstrates that PAL can efficiently protect dopaminergic neurons against neurotoxin injury in vitro and in vivo, and that the potential mechanisms may be related to its effects in increasing DJ-1, decreasing α-synuclein and its growth-promoting effect on spine density.

Baicalein prevents 6-hydroxydopamine-induced mitochondrial dysfunction in SH-SY5Y cells via inhibition of mitochondrial oxidation and up-regulation of DJ-1 protein expression.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by progressive loss of dopaminergic (DA) neurons at the substantia nigra. Mitochondrial dysfunction is involved in the mechanism of cell damage in Parkinson's disease (PD). 6-Hydroxydopamine (6-OHDA) is a dopamine analog which specifically damages dopaminergic neurons. Baicalein has been previously reported to have potential in the treatment of PD. The purpose of the present study was to investigate the mechanism of action of baicalein against 6-OHDA injury in SH-SY5Y cells. The results showed that baicalein significantly alleviated alterations of mitochondrial redox activity and mitochondrial membrane potential induced by 6-OHDA in a dose-dependent manner in SH-SY5Y cells compared with vehicle group. Futhermore, baicalein decreased the production of ROS and upregulated the DJ-1 protein expression in SH-SY5Y cells. In addition, baicalein also inhibited ROS production and lipid peroxidation (IC50 = 6.32 ± 0.03 µM) in rat brain mitochondia. In summary, the underlying mechanisms of baicalein against 6-OHDA-induced mitochondrial dysfunction may involve inhibition of mitochondrial oxidation and upregulation of DJ-1 protein expression.

Treatment responses of procaterol and CD38 inhibitors in an ozone-induced airway hyperresponsiveness mice model.

Airway hyperresponsiveness (AHR) and airway inflammation are key pathophysiological features of many respiratory diseases, such as asthma and chronic obstructive pulmonary disease (COPD). To evaluate the treatment responses of procaterol and CD38 inhibitors in an ozone-induced AHR mice model, we hypothesized that procaterol and two synthetic CD38 inhibitors (Compounds T and H) might have therapeutic effects on the ozone-induced AHR mice model, and the nuclear factor-kappaB (NF-κB) pathway and the CD38 enzymatic activity might be involved in the mechanisms. With the exception of the Control group, ozone exposure was used to establish an AHR model. Male Kunming mice in the Procaterol and CD38 inhibitors groups were treated with an emulsifier of procaterol hydrochloride, Compound T or H. Results indicated that (1) no drug showed severe toxicity in this study; (2) ozone exposure induced airway inflammation and AHR; (3) intragastric treatment with procaterol and Compound T achieved potent therapeutic effects, but Compound H did not show any therapeutic effect; (4) the NF-κB pathway was involved in both the pathogenic mechanisms of ozone and therapeutic mechanisms of procaterol and Compound T; (5) however, the in vivo effect of Compound T was not caused by its inhibitory activity on CD38. Taken together, procaterol and Compound T are potentially good drugs to treat asthma and COPD complicated with ozone exposure.

DJ-1 protein protects dopaminergic neurons against 6-OHDA/MG-132-induced neurotoxicity in rats.

Parkinson disease (PD) is the second most common neurodegenerative disease, and it cannot be completely cured by current medications. In this study, DJ-1 protein was administrated into medial forebrain bundle of PD model rats those had been microinjected with 6-hydroxydopamine (6-OHDA) or MG-132. We found that DJ-1 protein could reduce apomorphine-induced rotations, inhibit reduction of dopamine contents and tyrosine hydroxylase levels in the striatum, and decrease dopaminergic neuron death in the substantia nigra. In 6-OHDA lesioned rats, uncoupling protein-4, uncoupling protein-5 and superoxide dismutase-2 (SOD2) mRNA and SOD2 protein were increased when DJ-1 protein was co-injected. Simultaneously, administration of DJ-1 protein reduced α-synuclein and hypoxia-inducible factor 1α mRNA and α-synuclein protein in MG-132 lesioned rats. Therefore, DJ-1 protein protected dopaminergic neurons in two PD model rats by increasing antioxidant capacity and inhibiting α-synuclein expression.

Down-regulation of DJ-1 protein in the ejaculated spermatozoa from Chinese asthenozoospermia patients.

OBJECTIVE: To determine whether the expression of DJ-1 protein, whose levels in spermatozoa have been reported to be highly correlated with male infertility caused by toxicants, is changed in spermatozoa of Chinese asthenozoospermia patients. DESIGN: DJ-1 measurement by Western blotting, quantitive ELISA, and isoelectric-focusing electrophoresis (IFE) combined with immunoblotting. SETTING: Academic medical center and research laboratories. PATIENT(S): Asthenozoospermia patients (n = 113), including mild asthenozoospermia patients (n = 70) and moderate asthenozoospermia patients (n = 43), and age-matched control subjects (n = 58). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): DJ-1 in spermatozoa was determined by Western blotting and ELISA, the isoelectric point (pI) of DJ-1 by IFE combined with immunoblotting, and sperm superoxide dismutase (SOD) activity by an assay kit. RESULT(S): The sperm DJ-1 concentration in moderate asthenozoospermia patients was lower than those in mild asthenozoospermia patients and control subjects. DJ-1 with a more acidic pI was increased in asthenozoospermia patients. Sperm SOD activity was decreased in asthenozoospermia patients. CONCLUSION(S): DJ-1 levels are reduced in moderate asthenozoospermia patients. DJ-1 concentration is positively correlated with sperm motility and sperm SOD activity indicated by partial correlation analysis.

Design, synthesis and biological characterization of novel inhibitors of CD38.

Human CD38 is a novel multi-functional protein that acts not only as an antigen for B-lymphocyte activation, but also as an enzyme catalyzing the synthesis of a Ca(2+) messenger molecule, cyclic ADP-ribose, from NAD(+). It is well established that this novel Ca(2+) signaling enzyme is responsible for regulating a wide range of physiological functions. Based on the crystal structure of the CD38/NAD(+) complex, we synthesized a series of simplified N-substituted nicotinamide derivatives (Compound 1-14). A number of these compounds exhibited moderate inhibition of the NAD(+) utilizing activity of CD38, with Compound 4 showing the highest potency. The crystal structure of CD38/Compound 4 complex and computer simulation of Compound 7 docking to CD38 show a significant role of the nicotinamide moiety and the distal aromatic group of the compounds for substrate recognition by the active site of CD38. Biologically, we showed that both Compounds 4 and 7 effectively relaxed the agonist-induced contraction of muscle preparations from rats and guinea pigs. This study is a rational design of inhibitors for CD38 that exhibit important physiological effects, and can serve as a model for future drug development.

Proteome analysis of the left ventricle in the vitamin D3 and nicotine-induced rat vascular calcification model.

Vitamin D3 and nicotine (VDN) serve as an animal model of arterial calcification. The vascular calcification induced by the VDN model is always accompanied by compensatory left ventricular (LV) hypertrophy and impaired cardiac performance. To determine the possible mechanisms that are responsible for the effects of VDN on the LV, a 2-DE based proteomics approach was used to evaluate the changes in protein expression of the left ventricle in VDN rats, to our knowledge, for the first time. We identified sixteen proteins that were markedly altered and involved in mitochondrial function, heat shock protein activity, myocyte cytoskeleton composition and enzyme activity for energy metabolism. We describe, for the first time, a novel pathway (NDPK) that is involved in LV hypertrophy and enzyme activities of three of the sixteen clinical identified proteins: lactate dehydrogenase (LDH), SOD [Mn] and GST.

DJ-1-Mediated protective effect of protocatechuic aldehyde against oxidative stress in SH-SY5Y cells.

DJ-1 was identified as a causal gene for a familial form of early onset Parkinson's disease (PD), park 7. DJ-1 plays roles in transcriptional regulation and the anti-oxidative stress reaction. In this study, we found that protocatechuic aldehyde (PAL), a traditional Chinese medicine compound, bound to DJ-1 in vitro and that PAL protected SH-SY5Y cells but not DJ-1-knockdown SH-SY5Y cells from oxidative stress-induced cell death, indicating that the protective effect of PAL is mediated by DJ-1. Furthermore, PAL inhibited production of reactive oxygen species and the inhibition was abated in DJ-1-knockdown cells. PAL increased and decreased phosphorylation of AKT and PTEN, respectively, in SH-SY5Y cells, suggesting that the AKT pathway is one of the specific signaling pathways in PAL-induced neuroprotection. Moreover, PAL prevented superfluous oxidation of cysteine 106 of DJ-1, an essential amino acid for DJ-1's function. The present study demonstrates that PAL has potential neuroprotective effects through DJ-1.

3-O-demethylswertipunicoside protects against oxidative toxicity in PC12 cells.

Xanthone compounds have been reported to inhibit cancer cell growth as well as possessing antioxidant properties. The xanthone compound 3-O-demethylswertipunicoside (3-ODS), extracted from Swertia punicea HEMSL, has not previously been demonstrated to have clear neuroprotective effects. In our study, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell death assay revealed that treatment of PC12 cells with 3-ODS ameliorated the decreased cell viability induced by exposure to 1-methyl-4-phenylpyridinium ion (MPP+), rotenone or H2O2. The acridine orange/ethidium bromide (AO/EB) apoptosis assay demonstrated a significant suppression of cell death in PC12 cells. by 3-ODS treatment. 3-ODS increased the protein expression of both tyrosine hydroxylase (TH) and DJ-1 expression in PC12 cells. The current study demonstrates that 3-ODS has potential neuroprotective effects mediated via the elevation of TH and DJ-1 protein levels.

Neuroprotective xanthone glycosides from Swertia punicea.

Two new dimeric xanthone O-glycosides, puniceasides A (1) and B (2), a new trimeric O-glycoside, puniceaside C (3), and two new trimeric C-glycosides, puniceasides D (4) and E (5), together with 12 known xanthones were isolated from the entire plant of Swertia punicea. The structures of 1-5 were determined by HRESIMS and NMR spectroscopic methods. Compounds 2, 6, and 7 exhibited potent neuroprotective activity against H(2)O(2)-induced PC12 cell damage.

Protocatechuic acid inhibits rat pheochromocytoma cell damage induced by a dopaminergic neurotoxin.

Parkinson's disease (PD) is characterized by the progressive degeneration of dopaminergic neurons in the substantia nigra (SN) with the presence of alpha-synuclein inclusions termed Lewy bodies. The aggregation of alpha-synuclein into oligomeric species affects neuronal viability, having a causal role in the development of PD. The neuroprotective effects of protocatechuic acid (PAc) have been reported. However, the effects of PAc on tyrosine hydroxylase (TH) and alpha-synuclein in rat pheochromocytoma (PC12) cells treated with 1-methyl-4-phenylpyridinium ion (MPP(+)) remains unclear. In this study, we demonstrated that PAc inhibited the cytotoxicity, apoptotic morphology, reduction of TH expression and abnormal oligomeration of alpha-synuclein in PC12 cells treated with MPP(+). Taken together, our results indicate that the neuroprotective effects of PAc on PC12 cells treated with MPP(+) is related to the inhibition of the oligomerization of alpha-synuclein.

Kaempferol derivatives prevent oxidative stress-induced cell death in a DJ-1-dependent manner.

DJ-1, a causative gene product of a familial form of Parkinson's disease (PD), PARK7, plays a role in anti-oxidative stress, and loss of its function is thought to result in the onset of PD. Superfluous oxidation of cysteine at amino acid 106 (C106) of DJ-1 renders DJ-1 inactive, and such oxidized DJ-1 was observed in patients with the sporadic form of PD. In this study, we examined the relationship between DJ-1 and compounds extracted from traditional Chinese medicines possessing anti-oxidant activity. Of the 12 compounds tested, 5 were found to specifically bind to the C106 region by using a quartz crystal microbalance. Although 4 compounds prevented rat PC12 and primary neuronal cells from undergoing H2O2-induced cell death, the protective activity of 2 compounds, kaempferol 3-O-beta-rutinoside and 6-hydroxykaempferol 3,6-di-O-beta-D-glucoside, was diminished in cells transfected with siRNA targeting DJ-1, indicating DJ-1-dependent reaction of these compounds. Furthermore, these compounds reduced the level of reactive oxygen species and restored tyrosine hydroxylase activity that had been induced and compromised, respectively, by treatment of cells with H2O2. The results suggest that these compounds are useful lead compounds for PD therapy.

Protective effect of verbascoside on 1-methyl-4-phenylpyridinium ion-induced neurotoxicity in PC12 cells.

The neuroprotective effects of verbascoside, one of phenylpropanoid glucoside isolated from the Chinese herbal medicine Buddleja officinalis Maxim, on 1-methyl-4-phenylpyridinium ion (MPP(+)) induced apoptosis and oxidative stress in PC12 neuronal cells were investigated. Treatment of PC12 cells with MPP(+) for 48 h induced apoptotic death as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry, the activation of caspase-3 measured by the caspase-3 activity assay kit, the reduction in mitochondrial membrane potential with laser scanning confocal microscopy and the increase in the extracellular hydrogen peroxide level. Simultaneous treatment with verbascoside markedly attenuated MPP(+)-induced apoptotic death, increased extracellular hydrogen peroxide level, the activation of caspase-3 and the collapse of mitochondrial membrane potential. These results strongly indicate that verbascoside may provide a useful therapeutic strategy for the treatment of oxidative stress-induced neurodegenerative disease such as Parkinson's disease.